P95--LINKING DSB REPAIR AND CELL CYCLE CHECKPOINTS

P95——连接 DSB 修复和细胞周期检查点

• 批准号: 6386489
• 负责人: John HJ Petrini
• 金额: $ 17.46万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386489
• 关键词: DNA damage; DNA repair; cell growth regulation; chromosome disorders; gene targeting; genetic models; genetically modified animals; laboratory mouse; model design /development; phenotype; radiation genetics; radiation sensitivity; syndrome

Nijmegen Breakage Syndrome (NBS) is an autosomal recessive disorder characterized by marked cancer predisposition, sensitivity to ionizing radiation, and defects in DNA damage dependent cell cycle checkpoints. Because the cellular phenotypes of NBS strongly resemble those of cells established from ataxia telangiectasia (AT) patients, this syndrome has been described as an AT variant syndrome although it is genetically distinct. We showed that NBS is attributable to deficiency in an intrinsic member of the hMre11/hRad50 protein complex, referred to herein as p95. We have established that this complex, which is uniformly distributed in the nucleus prior to irradiation, relocalizes to sites of DNA damage in the nuclei of cells treated with ionizing radiation, and is restored to uniform distribution upon completion of DNA repair. The association of the complex with DNA damage, in conjunction with the finding that cellular responses to ionizing radiation are abrogated by p95 deficiency lead to the hypothesis that the complex acts to sense and signal the presence of DNA damage in irradiated cells. In this proposal, we will examine the function of p95 to address this hypothesis, and to elucidate its potential role the repair of ionizing radiation-induced DNA damage as part of the Mre11/Rad50 complex. We propose to carry out phenotypic analyses of p95 deficient cells expressing variants of p95 to define functionally important domains of the protein. To examine phenotypes that are not assayable in cultured cells and to assess genetic interactions of p95, p95 deficient mice will be constructed. Of particular interest in this regard are the genetic and functional interactions between p95 and ATM which will be assessed by breeding of p95 and ATM deficient mice. These studies will provide important information regarding the relative contributions of p95 and ATM in the cellular and organismal responses to ionizing radiation. In this proposal, p95 is examined from three complementary perspectives. First, at the cellular level, second, at the molecular level, and third, in vivo through the derivation of a munbs1 mutant mouse.

奈梅根断裂综合征(NBS)是一种常染色体隐性遗传病，其特征是明显的癌症易感性，对电离辐射敏感，以及DNA损伤依赖的细胞周期检查点缺陷。由于NBS的细胞表型与共济失调毛细血管扩张症(AT)患者建立的细胞表型非常相似，这种综合征被描述为AT变异综合征，尽管它在遗传上是不同的。我们发现NBS是由于hMre11/hRad50蛋白复合体的一个内在成员缺乏所致，这里称为P95。我们已经证实，这种在照射前均匀分布在细胞核中的复合体，重新定位到经电离辐射处理的细胞核中的DNA损伤部位，并在DNA修复完成后恢复到均匀分布。该复合体与DNA损伤的关联，以及P95缺乏使细胞对电离辐射的反应被取消的发现，导致了该复合体的作用是感知并发出辐射细胞中存在DNA损伤的信号的假说。在这个提案中，我们将研究P95的功能来解决这一假说，并阐明其作为Mre11/Rad50复合体的一部分在电离辐射诱导的DNA损伤修复中的潜在作用。我们建议对表达P95变体的P95缺陷细胞进行表型分析，以确定该蛋白的重要功能结构域。为了检测在培养细胞中无法分析的表型，并评估P95基因缺陷小鼠的遗传交互作用，将构建P95基因缺陷小鼠。在这方面特别感兴趣的是P95和ATM之间的遗传和功能相互作用，这将通过培育P95和ATM缺陷小鼠来评估。这些研究将提供有关P95和ATM在细胞和组织对电离辐射的反应中的相对贡献的重要信息。在本提案中，从三个互补的角度审查了P95。第一，在细胞水平上，第二，在分子水平上，第三，在体内，通过衍生出一种munbs1突变小鼠。